Several publications are referenced in this application by author name and year of publication in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. Several patent documents are also referenced to facilitate the practice of the present invention. The disclosure of each of these publications and patent documents is incorporated by reference herein.
Plants can respond to infection by microbial pathogens through the activation of a variety of defense responses. At the sites of infection, a hypersensitive response (HR) is often initiated. The hallmark of this response is the formation of necrotic lesions, a process that is likely due to programmed host cell death. In addition, associated with the HR is the restriction of growth and spread of pathogens. Frequently, defense responses are also activated in tissues distal to the sites of infection according to a phenomenon known as systemic acquired resistance (SAR). Development of SAR results in an enhanced and long-lasting resistance to secondary challenge by the same or even unrelated pathogens. Associated both with HR (and associated local resistance) and with SAR is the expression of pathogenesis-related (PR) genes, several of whose products have been shown to have antimicrobial activity (for review, see Klessig and Malamy, 1994; Hammond-Kosach and Jones, 1996; Durner et al., 1997; Dempsey et al., 1999).
A mounting body of evidence indicates that salicylic acid (SA) plays a key role in the activation of certain defense responses in a number of dicotyledonous species. For example, rises in endogenous SA levels correlate with the induction of PR genes and development of resistance in tobacco and cucumber (Malamy et al., 1990 and 1992, Metraux et al., 1990; Rasmussen et al., 1991). In addition, several mutants of Arabidopsis (e.g., cpr's, lsd's, acd's) have been isolated which constitutively express PR genes and show enhanced resistance. They also demonstrate elevated levels of SA (Bowling et al., 1994; Dietrich et al., 1994; Greenberg et al., 1994; Yoshioka et al., 2001). Conversely, Arabidopsis mutants defective in SA signal transduction (e.g., npr1, nim1, sai1) exhibit enhanced susceptibility to pathogens (Cao et al., 1994; Delaney et al., 1995; Shah et al., 1997). Exogenously applied SA also induces PR gene expression and enhanced resistance in tobacco (White, 1979; Antoniw and White, 1980) and a variety of other plants (for review, see Klessig and Malamy, 1994; Durner et al., 1997). Furthermore, transgenic Arabidopsis and tobacco plants that express the bacterial salicylate hydroxylase (nahG) gene, whose product converts SA into biologically inactive catechol, fail to develop SAR and show increased susceptibility to primary infections by both virulent and avirulent pathogens (Gaffney et al., 1993; Delaney et al., 1994).
During the past several years, attempts to elucidate the mechanisms of SA action in plant disease resistance have been made by identifying the cellular components with which SA interacts. Initial studies led to the identification of a SA-binding protein, SABP, that was later shown to be a catalase (CAT) (Chen and Klessig, 1991; Chen et al., 1993a). Further analysis demonstrated that SA inhibited tobacco catalase activity in suspension cells and in crude leaf extracts. SA also inhibited the purified enzyme (Chen et al., 1993b; Conrath et al., 1995; Durner and Klessig, 1996). Thus, it was proposed that increases in SA after pathogen infection might inhibit catalase activity, producing elevated levels of H2O2 that could activate certain defense responses, including PR gene expression. Supporting this hypothesis was the observation that prooxidants induced PR-1 gene expression (Chen et al., 1993b), while antioxidants suppressed the SA-mediated expression of PR-1 genes (Conrath et al., 1995; Wendehenne et al., 1998). In addition, the other major H202-scavenging enzyme, ascorbate peroxidase (APX), was subsequently shown to be inhibited by SA (Durner and Klessig, 1995).
In contrast, several other studies have questioned the role of H2O2 and the SA-mediated inhibition of CAT and APX during the activation of defense responses. No detectable increases in H2O2 levels were found during the establishment of SAR (Neuenschwander et al., 1995) and significant reductions in CAT activity were not observed in tobacco infected with Pseudomonas syringae or in leaf discs pretreated with SA (Bi et al., 1995). In addition, H2O2 and H2O2-inducing chemicals were unable to induce PR-1 gene expression in NahG transgenic plants (Bi et al., 1995; Neuenschwander et al., 1995). Moreover, high concentrations of H2O2 (150 mM–1000 mM) were shown to induce SA accumulation (Neuenschwander et al., 1995; León et al., 1995; Summermatter et al., 1995). Finally, transgenic plants having significantly lower CAT activity via transformation with CAT antisense or cosuppressing sense constructs, did not exhibit constitutive PR-1 gene expression unless there was concurrent development of necrosis (Chamnonpol et al., 1996; Takahashi et al., 1997). From these results, it appears that H2O2 acts upstream of SA in the signal transduction cascade rather than, or in addition to, acting downstream of SA.
One possible explanation for these conflicting findings is that SA and H2O2 are involved in a self-amplifying feedback loop (Van Camp, 1998; Draper, 1997; Shirasu et al., 1997). An alternative mechanism through which SA-mediated inhibition of CAT may signal defense responses is via the generation of SA free radicals, which could then activate a lipid peroxidation pathway (Durner and Klessig, 1996, Anderson et al., 1998). However, this possibility remains a topic of debate (Tenhaken and Rübel, 1997).
Taken together, these studies suggest that the activation of defense responses is mediated through the interaction of SA with other cellular factors, rather than, or in addition to interactions with CAT and APX. Thus, the isolation and identification of additional cellular factors associated with SA action in plant disease resistance is essential for elucidating the molecular mechanisms directing SA-mediated defense responses.